title: Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria.
abstract: Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed uniformly throughout the cell, demonstrating that Cht1 signal peptide functioned. In addition, thanatin(S) and thanatin(S)-FLAG chemically synthesized have both in vitro antimicrobial activities against P. syringae pv. tomato and B. cinerea. So, thanatin(S) is an ideal candidate AMPs for the construction of transgenic crops endowed with a broad-spectrum resistance to phytopathogens and the strategy is feasible to link a signal peptide to the target gene.
authors.affiliation: Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China.
authors.forename: Tingquan
authors.initials: T
authors.lastname: Wu
authors.forename: Dingzhong
authors.initials: D
authors.lastname: Tang
authors.forename: Weida
authors.initials: W
authors.lastname: Chen
authors.forename: Hexun
authors.initials: H
authors.lastname: Huang
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Yongfang
authors.initials: Y
authors.lastname: Chen
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis*; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D020171:Botrytis / Q000502:physiology; D002688:Chitinases / Q000737:chemistry; D060467:Disease Resistance; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D054884:Host-Pathogen Interactions; D019476:Insect Proteins / Q000096:biosynthesis*; D008856:Microscopy, Fluorescence; D012275:Oryza / Q000201:enzymology; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D010940:Plant Proteins / Q000737:chemistry; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D021382:Protein Sorting Signals; D044224:Pseudomonas syringae / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013172:Spores, Fungal / Q000502:physiology
chemical_list: D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C098076:thanatin; D049452:Green Fluorescent Proteins; D002688:Chitinases
keywords: AMP; Antimicrobial peptide; B. cinerea; Botrytis cinerea; G. cichoracearum; GFP; Golovinomyces cichoracearum; L; Phytopathogenic fungi and bacteria; Pseudomonas syringae pv. tomato; Pto; SP; Thanatin(S); antimicrobial peptide; day-postinoculation; dpi; green fluorescent protein; pL; pad4 transgenic line; signal peptide; transgenic line